Evaluation of the genomic DNA extracted from formalin-fixed, paraffin-embedded oral samples archived for the past 40-years

Abstract

y key words unitermos abstract Background: The most common human archival specimens are formalin-fixed, paraffin-embedded tissues (PETs). DNA can be extracted from PETs, but sometimes, it is unsuitable for molecular techniques as slow degradation of DNA occurs with time. Objective: The aim of this study was to verify and discuss if samples of oral PETs archived for the past 40-years are possible substrates for molecular biology studies, using PCR. Methods: The samples were submitted to phenol-chloroform extraction method. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis and amplification by PCR. Results: It was observed a weak positive correlation between genomic DNA yield and specimen age. The agarose gel electrophoresis demonstrated that genomic DNA length was more frequently composed of small fragments. The 268-bp fragments of the -globin gene was amplified in 55% of cases and preferentially in more recent ones, which showed strong amplification if compared with older samples. WAF1 gene with 149-bp presented weak but detectable amplification in 75% of cases. The 536-bp fragment of -globin gene was detected in 25% of samples. The amplification was intense in genomic DNA extracted from recent cases and weak in older ones. Conclusion: This study shown that, despite degradation, it is possible to use genomic DNA obtained from PETs, archived for the past forty years, in PCR amplification of small DNA products, being large DNA fragments more difficult to amplificate. Tecido parafinado DNA PCR Formol J Bras Patol Med Lab • v. 41 • n. 6 • p. 405-10 • dezembro 2005 ARTIGO ORIGINAL ORIGINAL PAPER Primeira submissão em 10/06/05 Última submissão em 15/12/05 Aceito para publicação em 15/12/05 Publicado em 20/12/05

No evidence of human papillomaviruses in non-genital seborrheic keratosis

“…Also, the negative control (in which we added sterile purified water instead of sample DNA) was negative by the HPVs PCR. (we took great care during the experiments to prevent cross-contamination; all negative controls returned negative results); (3) Moreover, as short DNA fragments are useful as a template for PCR,[15] GP5+/6+ primer sets were chosen to increase the success rate. (The most widely used PCR methods for the detection of HPV types are based on MY09/11 (yielding a 450 bp fragment) and GP5+/6+ (yielding a 142 bp fragment) primer sets.…”

Standardization ofTP53gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues

“…Recent works confirm the efficiency of this DNA extraction method (2,4,5,25,39) . The literature describes other methodologies for DNA purification, like Chelex ® , magnetic beads and home-made methods by digestion with proteinase K followed by extraction with phenolchloroform solution, also attaining good results (12,24,20,22,27,30) . Through this methodology, 100 μl DNA solution with average concentration of 119.73 ng/μl was obtained, what resembles the results found in the literature (25,39) .…”

“…In a study with samples preserved in paraffin and archived for a maximum time of 40 years, Libório et al (24) demonstrated that even though degraded, it is possible to obtain amplifiable DNA by PCR since the region to be amplified generates fragments of approximately 200 pb.…”

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

“…However, the relative abundance of this target, which was used as control for DNA extraction, does not necessarily mean that the parasite DNA was not degraded during fi xation and conservation (27) . The length of time during which samples are maintained in paraffi n blocks is an important factor for DNA amplifi cation, as previous research indicates that there is a negative correlation between the duration of storage and PCR success (25) . In the present study, while storage duration was not a limiting factor for the amplifi cation of the human actin gene, it could have had deleterious effects on the detection of Leishmania spp.…”